EMDOGAIW REGULATION OF CELLULAR DlFFERENTlATlON IN WOUNDEO RAT PERIODONTIUM

Laura Chano

A thesis submitted in wnfonnity with the requirernents for the degree of Master of Science Graduate Department of Dentistry

University of Toronto

@Copyright by Laura Chano 2001

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A bstract

EmdogairND regulation of cellular differentiation in wounded rat periodontium, by Laura Chano. Degree: Master of Science, Department of Periodontology, Faculty of Dentistry, University of Toronto, 2001.

EmdogainO is an enamel matrix derivative that may promote periodontal

regeneration by recapitulating critical events in tooth morphogenesis. I

hypothesized that EmdogainQY facilitates periodontal regeneration by promoting

the differentiation of cells required for the synthesis of periodontal ligament, bone

and cementum. Cell differentiation was examined in a rat periodontal window

wound model. Defects were filled with vehicle control or ErndogairNB (3 mg/ml or

30 mg/ml). Rats were sacrificed at 7, 14 and 21 days after wounding. Specimens

of periodontium were prepared for irnmunohistochemistry, morphometry and

radioautography. Rats treated with Emdogaim (30 mg/ml) showed widening of

the periodontal ligament at 7 days; by 14 and 21 days, periodontal ligament width

was restored to normal values for al1 groups. Emdogain exerted no effect on

cernentum thickness, bone volume, osteoid deposition rates, or extracellular

staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage

of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein

was unaffected by Emdogaim. Staining for a-smooth muscle actin

(myofibroblast marker) was abundant in the repopulating wound but was also

unaffected by EmdogaiMl. I conclude that EmdogaiM does not affect cell

differentiation or bone matrix protein synthesis in the repopulation response of

wounded rat molar periodontium .

Ackno wledgemenfs

I could not have accomplished this project without my supervisor, Dr. Chnstopher

McCulloch. I am grateful for the intelledual insight, encouragement, extreme

generosity and fnendship he has given me. I am also thankful for the technical

assistance of the laboratory staff, Hong Hong Chen and Balram Sukhu.

I would like to thank my family for al1 the attention, support,

encouragement, and love they gave me ovet the years. I am etemally grateful to

my mother for I tnily tealize and appreciate how much she has given and

supported me al1 through my Me. I also appreciate the encouragement that

Brenda gave me durhg these last three years and I deeply cherish the love and

support that James has given me.

Table of Contents

Abstract Acknowledgements Table List of Figures List of Abbreviations

ii iii vi vii viii

1. Literature Review 1

A. Penodontal diseases 1

B. Repair and regeneration of the periodontium 4

1. Wound healing in the periodontium 4

2. Cells in the periodontal wound healing response 7

3. Cellular markers of differentiation 10

4. Regulation of differentiation 12

5. Treatment approaches to regulate cellular differentiation 14

Cm Tooth and mot fornation 16

D. Emdogain 19

E- Mode1 systems and rationale 24

1. Non-human primates 26

2. Dogs

3. Rodents

II. Staternent of the problem 29

III. Materials and Methods

A. Wound mode1

B. Pmparation of implants and experimen fa1 design

C. Tissue preparation

D. Immunohistochemistry

E. Radioautography

F. Motphometric a n a l p s

G. Statistical analyses

IV. Results

A. Periodonfal ligament homeostasis

B. Cementum

C. Bone

D. Osteogenic difFerentiation

E. Fibmblast differentiation

F. Matrix formation

V. Discussion

A. Mode1

B. Mat& synthesis

C. DMemntiation

VI. Conclusions

VIL References

Table

Table 1. Matnx formation (appositional rate of osteoid and incorporation of

radiolabeled proline into nascent matrix proteins).

List of figures

Figure 1 A: Immunohistochemical staining for osteopontin

Figure 1 B: Morphometric analyses of periodontal ligament width

Figure 1 C: lmmunohistochemical staining for bone sialoprotein

Figure 1 D: Morphometric analyses of œmentum thickness

Figure 1 E: lmmunohistochemical staining for osteopontin

Figure 1 F: Morphometric analyses of nascent bone formation Figure 2A: lmmunohistochemical staining for intracellular osteopontin

Figure 28: Percentage of osteopontin cellsltotal cell count

Figure 2C: lmmunohistochemical staining for extracellular osteopontin

Figure 2D: Percentage of osteopontin stained matrix

Figure 3A: Immunohistochemical staining for intracellular bone sialoprotein

Figure 38: Percentage of bone sialoprotein cells/total cell wunt

Figure 3C: lmmunohistochemical staining for extracellular bone sialoprotein

Figure 3D: Percentage of bone sialoprotein stained rnatrix

Figure 4A: lmmunohistochemical staining for intracellular osteocalcin

Figure 48: Percentage of osteocalcin cellsRotal cell count

Figure 4C: lmmunohistochemical staining for extracellular osteocalcin

Figure 4D: Percentage of osteocalcin stained matrix

Figure SA: lmmunohistochemical staining for intracellular a-smooth muscle actin

Figure 58: Percentage of a-smooth muscle actin cellsltotal cell count

vii

Abbrevia fions

a-SMA

BMP

BSP

FGF

IGF

OC

OPN

PBS

PDGF

PL

TGF

Alpha-smooth muscle adin

Bone morphogenetic protein

Bone sialoprotein

Fibroblast growth factor

lnsulin growth factor

Osteocalcin

Osteopontin

Phosphate buffer saline

Platelet derived growth factor

Periodontal ligament

Transfonning growth fador

1. Literature Review

A. Penodontal diseases

Periodontal diseases are high prevalence infections of the periodontium that are

classified into two major groups- gingivitis and periodontitis (Page and

Schroeder, 1976). Gingivitis is a reversible inflammatory lesion of the

dentogingival junction that is not associated with bone loss (Lbe et al., 1965). In

wntrast, periodontitis is an ineversible destructive lesion (Page and Schroeder,

1976) of the connective tissue attachment and alveolar bone (Narayanan and

Page, 1983) that if left untreated can lead to tooth exfoliation.

Although root-borne, adherent bacterial biofilms are required for the

initiation of periodontal diseases, they are not sufficient alone to cause

progressive attachment loss (Offenbacher. 1996). Instead, epithelial and matrix

degradation are very likely attributable to disturbances in the balance between

host defense mechanisms and microbial assault. Accordingly, key factors in

disease progression indude both bacterial products (e.g. proteases, surface

membrane toxins) and host cell chernical mediators. Some of the host-derived

factors that are important in progressive periodontal tissue destruction include a

wide variety of pro-inflammatory molecules released by polymorphonuclear

leukocytes and macrophages and espedally the matrix metalloproteinases, zinc-

dependent endoproteinases which degrade collagen and other prominent

periodontal matrix proteins (Birkedal-Hansen et al., 1993). In bnef, the

development and progression of periodontitis is mnsidered to be a multifactorial

lesion involving interactions between the pathogenic wmponents of dental

biofilms, the vasculature, the innate and humoral immune systems, the epithelial

cells of the dentogingival jundion, stroma1 wnnective tissue cells and their

matrices (Offenbacher, 1996).

The reported prevalence (the fraction of a population exhibiting a

pathological condition at a specific time point) of periodontitis rnay Vary as a

result of the choice of measurement methods (Locker and Leake, 1993; Brown et

al., 1994). Nevertheless, a widely quoted estimate for the prevalence of

penodontitis in the United States is 36% (Brown et al., 1989). Notably, only a

fraction (i.e. 1 O-3O%) of those affected with periodontitis exhibit progressive

lesions and these susceptible individuals account for most of the disease burden

(Hirschfeld and Wasserrnan, 1978). As a result of the severity of the periodontitis

lesions in this susceptible group and because of intrinsic problems in treating

these infections, the dinical management of progressive penodontitis is a

substantial challenge to the dental health care system.

Numerous microbial species in dental biofilms have been implicated in the

initiation and progression of periodonti tis. However, only a relatively smal l

number have been examined in suffident detail to justify their inclusion as

putative periodontal pathogens. Some of the major pathogenic species include

Potphymnonas gingivalis, Actinobacillus actinomycetemcomitans and

Bactemides forsjdhus (Zambon, 1 996). In addition to the root-borne pathogen

load generated by these and other less virulent bacterial species, tome evidence

indicates that œmentum surfaces contaminated by periodontal pathogens

undergo morphological and biochernical changes that may contribute significantly

to the progression of the lesions (Aleo et al., 1974). Further, in the context of

periodontal wound healing, contaminated root œmentum may be a critical factor

in blocking reattachment of nascent collagen fibers to previously exposed root

surfaces since root-bound endotoxin can inhibit ceIl attachment in vitro (Aleo et

al., 1975).

Contamination of the root surface as well as the loss of potential growth

and differentiation factors from the cementum matrix are important obstacles for

periodontal repair and regeneration. Further, the mot surface is an avascular

structure and cannot directly contribute to the formation of blood vessels or to the

production of cells that are important for reattachment. Instead, the cells that

repopulate and attach to the exposed root surface must migrate from adjacent

vascularized wound edges. Hence, our understanding of root surface biology and

its role in cell recruitment and cell differentiation is pivotal in the regeneration of

perÏodontal tissues, particularîy since the repopulating cells in a periodontal

wound must migrate a considerable distance before they can contribute to

healing at the root surface. Notably, cells from contiguous endosteal spaces

(McCulloch et al., 1987) rnay be able to contribute to the repopulation of healing

periodontal wounds. However, we have limited ability to manipulate this

repopulation response favourably and predictably sinœ we do not know how to

selectively recniit or promote the differentiation of the cell types that are required

for nascent tissue synthesis (e.g. cementoblasts, osteoblasts, fibroblasts).

B. Repair and regeneration of the periodontium

1. Wound healing in the periodontium

Wound healing is the process by which an organism attempts to reconstitute

tissues damaged by injury and subsequently restores their function.

Reconstitution can be achieved either through regeneration or repair or both. In

regeneration the architecture and function of lost periodontal tissues are

completely renewed. When this is not accomplished, the damaged tissue is

repaired and the injured tissue is replaced with a fibrous scar or by cells and

attachment complexes that are not found in the original tissue structure (e.g. long

junctional epithelium). Repair is a biological process by which the continuity of

disrupted tissue is restored by new tissues that do not replicate the structure and

function of tissues destroyed by disease or injury (Arnerican Academy of

Periodontology, 1 992).

Following tissue injury, there is rapid formation of a blood clot and

stabilization by fibrin. Between 1 3 days following injury, polyrnorphonuclear

leukocytes and macrophages are reuuited to the wounded site. These cells

debride the wound site by phagocytosis, kill local microorganisms and facilitate

tissue remodeling by the release of matrix metalloproteinases and other neutral

proteases. The macrophage population also contributes an important regulatory

role since cytokines released by macrophages help to control local cell fundion.

Later on in wound healing (4-10 days), a provisional matrix is formed by

endothdial cells and fibroblasts that is the prewrsor for more mature periodontal

tissues. The granulation tissue f m e d at the wound site is colonized 4-10 days

after wounding by progenitor cells from the periodontal ligament and ccntiguous

endosteal spaces. The progenitors can undergo several rounds of ceIl division to

become periodontal ligament fibroblasts, cementoblasts and osteoblasts. In tum

these cells secrete the proteins of the specialized tissues of the periodontium

including periodontal ligament, cementum and bone. Finally, there is a long-terni

remodeling phase of the newly formed tissues that may require as long as 7

year.

The critical events in wound healing of many tissues and organs exhibit

several cornmon features and have been studied in depth using, most commonly,

experimental skin wound models. However, in cornparison to the relatively simple

wound healing responses that ocair in skin, healing of pefiodontium is

complicated by the diversity of the celi and tissue types as well as the inability of

the root surface to be vascularized. Notably, wound healing studies in the

periodontium demonstrate that the phenotype of the repopulating cells exert an

impact on the type of repair processes that occur subsequently (Nyman et al.,

1982). This finding and earlier work by Melcher (1 970) established the notion that

the tissue ongin of the cells impaded on the nature of the tissue that is ultimately

foned; this concept has provided the basis for the socalled guided tissue

regeneration procedure.

Early events in the wound healing process may have an impact on later

outcornes. Notably, Polson and Proye (1 983) extracted and re-implanted normal

teeth in squirrel monkeys after surgically denuding the coronal root surface of

connective tissue fibers and œmentum by root planing. Some test spedmens

were treated with topical application of citric acid; these teeth exhibited a different

response campared to control specirnens that were not acid-treated. In controis,

the epithelium migrated rapidly along the denuded root surface. Epithelium

reached the alveolar crest by 3 days and extended into the periodontal ligament

space ta the level of the denuded root by 21 days. In contrast, in roots treated

with citric acid, the epithelium did not migrate significantly along the denuded root

surfaces. At 1 and 3 days, infiammatory cells were embedded in a fibrin network

that was apparently attached to the root surface. At 7 and 21 days, the wound

site was repopulated with cannedive tissue cells and collagen fibers had

replaœd the fibrin. The authors speculated that formation of the collagen fiber

attachment to the root surface was preceded by a fibrin linkage. This linkage was

considered to be an important early event in the wound healing response and

underlines the importance of wound stabilization by fibrin. Further, the paper

highlights critical steps played by specific proteins (i.e. fibrin) in the wound

healing process.

2. Cells in the periodontal wound healing response

A very large vatiety of different cell types participate in periodontal wound healing

but only the synthetic cells of petiodontal tissue matrices will be described in any

detail here. Bnefly, the cells participating in wound healing originate from different

precursor populations. For example, polymorphonudear leukocytes are gtanular

leukocytes 7-9 Pm in diameter that are of hematogenous (myeloid) origin and

contribute important microbiocidal and phagocytic functions. Platelets are 2 4 pm

diarneter dis= devoid of a nudeus that are fomed from megakaryocytes and

play aitical roles in blood clotting. Some of the platelet granules also contain

important cytokines that regulate wound healing such as transforming growth

factor+ and platelet derived growth factor. The synthetic cells in periodontal

wounds are derivad from locally proliferating stroma1 precutsors and include

fibroblasts, cementoblasts and osteoblasts.

Fibroblasts are large, often flat, branching cells which appear fusifonn or

spindle-shaped in profile and exhibit an oval or elongated nucleus. They are

responsible for the formation and rernodelling of collagen fibers and are thought

to elaborate most, if not all, of the amorphous component of the rnatrix

(principally glycosaminoglycans). Osteoblasts have vanous shapes (i.e cuboidal

or squamous) depending on their synthetic activity. They exhibit a large nudeus

and are found on bone surfaces where matrix synthesis and minerakation are

extant. Cementoblasts are by definition, associated with the surfaces of newly

foming cementum and typically exhibit similar morphologies as osteoblasts.

However, 'restingn cernentoblasts are not squamous in shape but rather appear

to retract from the cementum margin and instead appear to be more fibroblastic

in appearance.

When considering the formative cells of the periodontal tissues, it is

worthwhile to consider their ability to divide and multiply since in wounds, cell

multiplication is critical for repopulation and reparative processes. In general,

mammalian cells may be classified into 3 types on the basis of their proliferative

potential (Leblond, 1 981 ). Renewal cell populations continuously divide and have

considerable division potential throughout postnatal Iife. For example, the crypt

and villus cells lining the gastrointestinal tract and the progenitor cells of blood

comprise renewal cell populations. Expanding cell populations are comprised of

cells that have limited division potential during the lifetime of the organism but

can divide extensively on demand, most notably after injury. Liver and kidney

cells are examples of expanding ceIl populations. Static or non-renewal cells are

cells which have exited the cell cycle, undergone differentiation, and do not

divide. Central nervous system neurons are examples of static cells; the deletion

of these cells by injury or loss of blood supply (Le. stroke) has important

implications for restoration of function.

From morphological and cell kinetic studies of mouse periodontal ligament

under conditions of both normal function and while undergoing regeneration,

several important properties of the formative cell population have k e n deduced.

First, the progenitor cells of periodontal ligament in paravascular zones exhibit

some of the features of stem cels of renewing populations. These cells

classically exhibit extensive proliferative capacity (Gould et al., 1977), are

responsive to control mechanisms (Gould et al., 1977, l98O), demonstrate self-

renewal capacity (Gould et a/., 1980) and are spanely distributed within the

proliferative compartment (McCulloch and Melcher, 1983a). Second, the progeny

of paravaswlar progenitor cells migrate to bon8 and cementum surfaces where

they differentiate into cementoblasts and osteoblasts (McCulloch and Melcher,

1983b; McCulloch et al., 1987). Third, the turnover of periodontal ligament cells is

relatively slow (McCulloch and Melcher 1983~). Collectively, these data suggest

that the formative cells of the periodontal ligament comprise as slowly renewing

cell population. Finally, it is possible that the relatively primitive cells immediately

surrounding blood vessels (Le. paravascular cells) are stem cells since they

exhibit many of the classical features of stem cells described above (McCulloch,

1 985).

In addition to providing cells for repopulation of the fibroblast population,

the periodontal ligament may also serve as a reservoir of precursor cells for

cementoblasts and osteoblasts. Cementoblasts are important for the apposition

of cementum that may be required in response to trauma and for the repair of

darnaged root surfaces (Beertsen and Everts, 1990). Periodontal ligament

fibroblasts may also have the ability to differentiate into cernentoblasts

responsible for the synthesis of acellular extrinsic fiber cementum (Beertsen and

Everts, 1990). Alveolar bone also undergoes continual remodeling under

physiological conditions and the progeny of paravascular fibroblastic cells in the

periodontal ligament c m migrate to the bone surface and there diffetentiate into

osteoblasts (McCulloch and Melcher, 1983a). Consequently, cells from the

periodontal ligament may be crucial in the formation and repair of cementum and

bone, processes that are important in maintaining the structural integrity of the

periodontium.

During tooth formation, cementoblasts and periodontal ligament fibroblasts

originate pnmarily from cells of the dental follicle proper and the cells of the

perifollicular mesenchyme respectively (Cho and Garant, 1988; 1989). Shortly

after the onset of cementogenesis, cementoblasts detach from the newly fonned

cementum surface and appear to join the fibroblast population in the periodontal

ligament. This suggests that both the dental follicle proper and perifollicular

mesenchymal cells contribute to the periodontal ligament cell pool. Thus the

periodontal ligament cell population is apparently a mixed cell population

containing precursors for cementogenic cells (among others). However, a more

definitive understanding of the wound healing potential of the formative cells of

the periodontium has (and will) rely on rnethods to measure unambiguously the

differentiation potential of the cells.

3. Cellular markers of d'ifferentiation

Molecular markers of cellular differentiation have been utilized in many studies

focusing on for example, blood formation (Till and McCulloch, 1980) and bone

formation (Bruder et al., 1990; Turksen et al., 1992). In mineralized tissue

formation several extracellular matrix macromolecules including osteopontin,

osteocalcin, and bone sialop rotein are expressed by differentiating osteogenic

cells. Measurement of these proteins has been used to identify discrete stages in

the formation of bone in vivo (Chen et al., 1992; Yoon et al., 1987) and for

studies of ceIl differentiation in periodontal tissues (Lekic et al.. 1996b).

Osteopontin and bone sialoprotein are major noncollagenous proteins

rscreted by osteoblastic cells and deposited into the bone matrix (Kasugai et al.,

1991 ; Nagata et a/. , 1 991 ). Both proteins are glycosylated, phosphorylated, and

sulfated and both have Arg-Gly-Arp (RGD) sequences that may provide cell

attachment motifs (Butler, 1989; Heinegard and Oldberg, 1989). However,

whereas bone sialoprotein is expressed almost exclusively by differentiated

mineralized tissue-forming cells (Bianco et al., 1991; Chen et al., t991a, 1992),

osteopontin is expressed by osteogenic and also nonosteogenic cells (Denhardt

and Guo, 1993). In osteogenesis, osteopontin mRNA is expressed during matrix

formation. In cornparison. the expression of bone sialoprotein coincides with

initial bone mineralization and is believed to be a nudeator of hydroxyapatite

crystal formation (Hunter and Goldberg, 1993). Since osteopontin and bone

sialoprotein are expressed in alveolar bone, cernentum and dentin (Chen et al.,

1991 a, 1993), the differential expression of these proteins can be used to study

the formation and repair of periodontal tissues and the contribution of periodontal

ligament cells to bone matrices that contain osteopontin and bone sialoprotein.

In camparison to osteopontin, bone sialoprotein (Kasugai et al., 1992) and

osteocalàn (Chen et al., 1992) are expressed at later stages of bone cell

differentiation and may be expressed also by cernentoblasts. Hence the

identification of these matrix proteins could suggest the presence of cells

committed to the osteogenic or cementogenic lineages. Osteocalcin is a y-

carboxyglutamic acid (Gla)-containing protein that is also a major

noncollagenous bone protein. It has been used for studies of later stages of bone

cell differentiation in rat tissues (McKee et al., 1993). Collectively, osteopontin,

bone sialoprotein and osteocalàn c m provide useful markers for bone and

possibly cementum cell differentiation.

a-smooth muscle actin is an actin isoform that has been extensively

studied in the differentiation of fibroblasts into myofibroblasts and their role as

contractile cells (Desmouliere et al., 1993). The a-smooth muscle actin isoform is

a well-descfibed fundional marker for a subpopulation of contractile periodontal

fibroblasts (Arora and McCulloch, 1994) and its assessrnent may facilitate the

identification of specific, periodontal cell subpopulations that are important in

rnatrix contraction and wound remodelling (Arora et al., 1999).

4. Regulation of differentiation

A wide variety of molecules participate in the regulatory processes required for

periodontal regeneration. Based on their mechanism of action, two important

classes of regulatory molecules are worth considering in the context of

periodontal regeneration and how exogenous application of these factors rnay be

exploited to facilitate regeneration: 1) growth factors and other inflammatory

mediators, including cytokines, lymphokines, and chemokines; 2) adhesian

molecules and matrix components such as fibronectin, laminin, collagens,

proteoglycans, and hyaluronan. The first group of molecules can regulate the

migration, proliferation and differentiation of cells during inflammation and wound

repair. Adhesion molecules localize cells at required sites and may be specific for

certain cell types or may be non-specific in their interactions. Matnx components

also provide important adhesive functions and are needed for the structural and

physiologic integrity of new tissues as well as for regulating cell differentiation.

These molecules may originate from the circulation or may be produœd locally

by cells residing in the tissue matrix.

Polypeptide growth factors are naturally ocuirring biological mediators

that orchestrate critical cellular events involved in regenerative processes sudi

as cell proliferation, chernotaxis, differentiation and matrix synthesis (Matsuda et

al., 1992). The growth factors found in bone matrices indude transfoming growth

factor-B (TGFS), insulin-like growth factors I and II (IGF-I and II), platelet-derived

growth factor (PDGF), acidic and basic growth factors (a- and b-FGF) and bone

morphogenetic proteins (Graves and Cochran, 1994). The prirnary cellular

sources of PDGF, FGF, TGF-f3 and IGF are platelets, macrophages and

osteoblasts (Giannobile, 1996). These factors are also stored in bone matrix and

may be released during bone remodelling, thus helping to couple bone formation

to resorption (Linkhart et al., 1996). In bone, osteoblasts are important target

cells of these growth factors although sorne of these cytokines can induce

periodontal ligament cells to proliferate as well (Graves and Cochran, 1994).

There are abundant epidemial growth factor (EGF)-binding sites on

differentiating perifollicular mesenchyme cells as well as on mature periodontal

ligament fibroblasts exhibiting synthetic adivity (Cho et al., 1991 ). This finding

suggests that EGF plays an important role in cell differentiation as well as during

the active synthetic activity of mature cells. Conversely, low binding of EGF to

dental follicle cells, precementoblasts and cementoblasts indicates that EGF

probably has little or no effect on cementoblast difFerentiation. Consequently, the

EGF-receptor may act to negatively regulate the differentiation of periodontal

ligament fibroblasts into mineralized tissue-forming cells (Cho et al., 1991 ).

5. Treatment approaches to regulate cellular differentiation

As noted above, vanous wound healing events and cellular activities assouated

with healing are regulated by polypeptide growth factors. Several exogenously

applied growth factors have been utilized to treat naturally and experimentally-

induœd periodontal defects in anirnals (Graves and Cochran, 1994). In beagle

dogs with naturally occurring periodontitis, Lynch et al. (1 989) demonstrated that

the combination of PDGF with IGF-1 stimulated regeneration of the periodontium,

possibly through its efFed on mesenchymal cells. Indeed, the formation of new

cementum-like deposits and alveolar bone was present in growth factor-treated

sites but not in mt ro ls receiving surgery and placebo gel.

Giannobile et al. (1 996) compared the effects of platelet-denved growth

factor-BB and insulin-like growth factor-1, individually and in combination, on

periodontal regeneration in Cynomolgus monkeys. Ligature-induced periodontitis

was initiated and after periodontal lesions were established, surgery was

performed, and either gel (i.0. vehicle control), or gel containing PDGF-BB, IGF-I

or bath was applied to the exposed root surfaces. They found that IGF-l alone, at

the dose tested, did not significantly alter periodontal healing. In contrast, PDGF-

86 alone strongly stimulated the formation of new attachment. In addition, the

PDGF-BBIIGF-I combination resulted in significant increases in new attachment

and production of new bone in the osseous defects 4 and 12 weeks post-

surgically. Further, in a study performed in monkeys, Rutherford et al. (1993)

demonstrated that a combination of PDGF and dexamethasone could also

promote regeneration.

Howell et al. (1997) perfomed a clinical trial to evaluate the therapeutic

effect of a combination of recombinant human PDGF-BB and recombinant

human IGF-l in patients with periodontitis. Subjects were treated in a splitmouth

design. The test sites received the local application of the dnig in one of two

doses while the control sites were either treated surgically only or received a

vehicle gel. Reentry procedures were perfomed 6 to 9 months post-surgically.

Patients treated with the higher dose of the wmbined dnig protocol

demonstrated statistically significant increases in alveolar bone formation. Sirnilar

results were found for furcation defeds.

Collectively, these studies suggest that the topical application of growth

factors has promise in the treatment of periodontitis but further studies are

needed to characterite the mechanism of action, the kinetics of dnig degradation

and dnig release, and to more carefully demonstrate the reliability and efficacy of

the therapeutic maneuver. While the topical application of these factors may

ultimately show promise clinically, there is uncertainty as to whether the cells

involved in wound healing in adult periodontal tissues are actually capable of

regeneration. Thus an important question is whether wound healing in aduît

tissues recapitulates the events of root and periodontal ligament formation that

occur during tooth development. Cognizant of these issues, in the next section I

will describe the salient events of tooth formation that relate to the development

and formation of the periodontal ligament and alveolar bone.

C. Tooth and mot fornation

In development, the organization of cells into tissues is accomplished in part

through morphogenesis; cellular diversification is achieved through the process

of differentiation. These two processes, in addition to growth and reproduction.

comprise critical components of mammalian development and are central to

organogenesis. After the 3 germ layers (ectoderm, mesodenn and endoderrn)

are formed through primary induction, organogenesis is initiated. The cells of the

germ layers interact and rearrange thernselves into specialized organs such as

the limbs, eyes and teeth. The formation of these and other complex organs

depends on sequential and reciprocal interactions between the epithelial and

mesenchyrnal tissue, a proœss called secondary induction.

Teeth arise as a result of secondary induction between the oral epithelium

and its adjacent dental mesenchyme (Lumsden, 1988; Thesleff and Sharpe,

1997). The oral epithelium originates from the stomodeal or pharyngeal region of

the developing embryo (Thesleff and Sharpe, 1997). The mesenchymal cells

underîying the epithelium of the first brachial arches (where the future rnaxillary

and mandibular processes reside) are of neural crest origin (Osumi-Yamashita et

al., 1994). Following migration of neural crest cells to the first brachial ara, a

group of neural crest cells interact with the overîying oral epithelium and cause

an invagination of a band of epithelial cells to form the dental lamina. Indeed, the

first distinct morphological feature indicative of tooth development is the

formation of the dental lamina and the localized thickening of the dental

epitheliurn. As the dental lamina expands, the epithelium invaginates into the

underiying mesenchyme, forming an epithelial bud (the bud stage of tooth

development). The mesenchymal cells condense around the bud and fonn the

dental papilla which gives rise to both the dental pulp and dentin-secreting

odontoblasts (Peters and Balling, 1 999). Notably, the proteoglycans syndewn-1

and tenascin, two important cell adhesion molecules, are involved in

mesenchymal cell condensation (Thesleff et ai., 1995). At the cap stage of

development, the enamel knot appears within the epithelial cornpartment mi le

the bel1 stage is characterizad by rapid cell proliferation and dental crown

formation (Kettunen and Thesleff, 1998).

The multiple redprocal interactions between oral epithelium and its

adjacent mesenchyme dernonstrate that the inductive and wmpetence

properties of each tissue are temporally and spatially restricted (Dassule and

McMahon, 1998). This has been shown in tissue recombination experiments in

which a non-dental, neural crest-derived mesenchyme from a mouse could be

induœd by an oral epithelium isolated from the mandibular arch. Apparently, the

odontogenic potential resides in the epithelial layer at this early stage of tooth

development. At the onset of tooth morphogenesis, odontogenic signaling events

originate fiom the dental epithelium (Lumsden, 1 988). The instructive signaling

center reverts to the enamel knot of the dental epithelium (Peters and Balling,

1999).

The inner enamel epithelium plays an essential role in the differentiation of

odontoblasts of the crown. For the development of root odontoblasts, the inner

epithelial cells of the root sheath are required. The detachment of the inner

epithelial cells from the dentin surface and the fragmentation of the underlying

basal lamina may result from the active migration of preœmentoblasts toward the

dentin surface and their penetration between the dentin surface and the inner

epithelial cells during their differentiation into cementoblasts (Cho and Garant,

1988). The disruption of the root sheath exposes the newly deposited dentin

matrix to the follicular connective tissue. a possible requirement for cementoblast

differentiation. The earliest sign of precementoblast differentiation is the

projection of cell processes from their leading edge toward and into the space

previously occupied by the root sheath. Cementoblast differentiation involves

directed cell migration of precementoblasts toward the dentin matrix causing

disruption of the epithelial root sheath and the eventual contact of newly

differentiated cementoblasts with root dentin. Upon contact with the dentin

surface, these cells exhibit the appearance of fully differentiated cementoblasts

involved in forrning acellular extrinsic fiber cementum. Differentiation and

migration of precementoblasts from the dental follide toward the dentinal surface

are probably initiated by a chemoattractant andhr promoted by an adhesion

gradient within the local extracellular dentinal matrix or from the inner basement

membrane of the root sheath (Cho and Garant. 1988). Soon after œmentum

deposition, newly differentiated cementoblasts detach and move away from the

newly fomed cementum, which implies that the signal for their chemotaxis is

transient. They then become a part of the periodontal ligament fibroblast

population (Cho and Garant, 1989).

The formation of acellular cementum involves an initial phase of directed

cell migration, followed by attachment and cementum matrix deposition during

which the cementoblast morphotype is deariy expressed. It then detaches and

assumes a fibroblast-like morphotype (Cho and Garant, 1989). The

mesenchymal cells of the dental follide undergo direded migration toward the

dentin and the perifolliwlar mesenchymal cells differentiate primarily into the

periodontal ligament fibroblasts. Thus both the dental follicle proper and

perifallicular mesenchyme contribute to the pool of periodontal ligament cells

(Cho and Garant, 1989).

Wth this brief background of tooth and periodontal ligament development

in mind, there has been considerable interest in applying principles of tooth

development to periodontal wound healing. Notably, the work of Lars

Hammarstrom has been pivotal in developing a biological basis for topical

application of enameldenved proteins that may be able to facilitate periodontal

wound healing. These enamelderived proteins are now commercially available

under the trade-name " E m d o g a i ~ .

D. Emdogalm

Prior to reviewing the putative role of EmdogainQD in periodontal regeneration, I

will first provide some of the relevant background that led to its dinical

development. Alrnost 20 years ago, it was suggested that the formation of new

periodontal attachment may be promoted selectively by 'guiding" periodontal

ligament cells into periodontal wounds (Nyman et al., 1982). It has yet to be

shown definitively that the cell exclusion methods described by Nyman and

colleagues in any way guide either tissue formation or cell migratory behaviour.

Indeed, while it has been suggested that the repopulation of wounded

periodontium with cells onginating from the periodontal ligament may result in

enhanced healing and functional repair (Boyko et al., 1981 ; Nyman et al., 1981;

Egelberg, 1987). lineage studies in which the origin of repopulating cells was

definitively demonstrated have not been conducted. Further, although it has been

suggested that only the cells located within the periodontal ligament have the

ability to regenerate the tissues of the periodontal attachment apparatus (Melcher

1976), it is equally likely that cells from contiguous endosteal spaces (McCulloch

et al., 1987) can also contribute to the repopulation of healing periodontal

wounds.

The metabolic behaviour of cells that originate from both PL and endosteal

spaces is modulated by factors (e.g.. chemokines and cytokines) that regulate

their reauitment from progenitors and may affect the outcornes of wound healing

(see above Section B). In this context, many factors including extracellular matrix

components (mg. collagen or enamel matrix proteins) may contribute to

enhanced regeneration. In this context, the major proteins of the enamel matrix

are known as arnelogenins. They are expressed at the apical end of the foming

toot (Lindskog 1 982a,b; Lindskog and Hammarstrorn, 1982) and comprise -90%

of the enamel matrix. The remaining 10% indude proline-rich enamel proteins

(Fukae and Tanabe, 1 987), tuftelin (Deutsch et al., 1 991 ), tuft proteins (Robinson

et al., 1975), various serum proteins and at least one salivary protein (Brookes et

al., 1995).

During root formation, Hertwig's epithelial root sheath, a derivative of the

inner cells of the enamel organ, induces the mesenchymal cells of the dental

papilla to fom the mantle predentin before it disintegrates and detaches from the

root surface. The mesenchymal cells exposed to the newly fomed dentin are

believed to induce cementogenesis (Cho and Garant, 1988). However, an

exposed dentin surface is thought to be an insuffident stimulus for œmentoblast

differentiation. Instead, it has been proposed that enamel matrix proteins may be

involved in the formation of acellular cementum during nascent root development

(Slavkin, 1976; Lindskog 1 982a, b; Slavkin et al., 1989, Hammarstrom, 1997). In

fact, there is increasing evidence that the inner epithelial cells of Hertwig's

epithelial root sheath may have the potential to produœ and secrete these

enamel-like proteins during root formation (Slavkin. 1976; Lindskog 1982a,b,

Slavkin et al., 1989, Hammarstrbm, 1 997). Further, during the initial formation of

the enamel matrix. mesenchyrnal cells exposed to these proteins differentiate

into cementoblasts. In addition, a non-cellular cementum-like tissue is formed on

the surface of enamel matrix when it is exposed to these mesenchymal cells

(Hammarstrom, 1997).

Slavkin et al. (1989) showed that acellular cementum contains proteins

that are immunologically related to proteins present in the enamel matrix. This

finding suggests that acellular œmentum is a secretory product of epithelium and

that it can only be fomed during tooth development. Formation of acellular

cernentum is unlikely during the adult Iife of mammals as the epithelial cells that

may regulate its formation (aside from the rests of Malasset) are no longer

present in the adult.

Alterations to œmentum structure and biochemical composition are

central factors in the periodontal disease proœss (Aleo et al., 1974; Robinson

1 975). Consequently, these changes impose significant limitations on

regenerative patential and impact on the management of the disease and

repairlregeneration treatment protocols. Hence, the application of enamel matrix

proteins to the exposed root surfaces could promote wound healing and the

regeneration of the periodontal ligament, cementum and alveolar bone. Based on

these observations, it has been hypothesized that enamel proteins play a

stimulatory role in the formation of cementum (Slavkin 1976; Hammarstrom et al.,

1 997).

Enamel matrix proteins have been relatively well conserved during

evolution (Slavkin and Diekwisch, 1996) and there appears to be a high degree

of amino acid similarity between the sequences of porcine and human enamel

proteins (Brookes, 1995). Arnelogenin is an enamel protein expressed at the

apical end of the forming root and is present in the area where cementogenesis

is initiated. An acellular cementum-like tissue is formed on the surface of enarnel

matrix when it is exposed to mesenchymal œfls of the dental follide. Thus

enamel matrix proteins are possibly involved in the development of œmentum.

With this in mind, enamel matrix derivatives have been developed as a dinical

treatment to promote periodontal regeneration. The commercial product,

EmdogairvID, has been described as a resorbable material and consists of

hydrophobic enamel matrix proteins bebnging to the amelogenin family extracted

from the enamel of developing teeth in porcine embryos (Heijl et al., 1997).

Emdogaim is supplied as freeze-âried enamel matrix proteins in a viscous

carrier, propylene glycol alginate. The carrier is a propylene glycol ester of alginic

acid (Gestrelius et al., 1997a).

The mechanism of action of enamel matrix derivative is not known in detail

but conceivably, the derivative mimics the role of enamel proteins in

cementogenesis during nascent root development. It appears that the temporary

deposition of enamel matrix proteins ont0 a root surface is an essential step

preceding the reformation of acellular cementum and that the formation of

periodontal ligament and alveolar bone is dependent on formation of acellular

cementum (HammarstrBm, 1997). The use of enamel proteins as an adjund in

periodontal surgery could possibly provide a 'natural" extracellular matrix for

recolonization of previously diseased mot surfaces by cells expressing a

cementoblastic phenotype.

Experimental studies have shown that aœllular cementum is fomed when

mesenchymal cells of the dental follicle are exposed to endogenous or

exogenous enamel matrix (Hammarstrom, 1997) so cementwn proteins cwld

modulate the biological activities of periodontal ligament and possibly gingival

fibroblasts. Indeed, Gestrelius et al. (1 997b) have demonstrated that periodontal

ligament cells show increased proliferation and mineralized nodule-formation in

the presence of these enamel proteins. Further, enamel matrix proteins may also

affect bacterial colonization of the root surfaces as the physico-chemical

properties of the environment (e.g. hydrophobicity) may modulate bacterial

adherence. Regardless of the favorable dinical results obtained in periodontal

therapy with EmdogainQB (Pontoriero et al., 1 999; Heden et al., 1 999; Sculean et

al., 1999), the biological effects and its mechanism of action are still unclear.

E. Mode1 sysfems and rationale

Elucidation of the critical regulatory factors in periodontal wound healing will likely

be predicated on the use of appropnate model systems, perhaps combining in

vitm as well as in vivo methods. In vitm cell culture analyses of periodontal cells

provide simplified approaches for understanding basic molecular mechanisms in

regenerative processes but without the interference of multiple cell types and

confounding in vivo factors (e.g. bacterial contamination). However, conclusions

from in vitro investigations may be incomplete since they cannot recapitulate the

events involved in regeneration and the cornplex intercellular communication

systems that may exist between the different types of periodontal œlls

(McCulloch, 1993). The major, and by far the most wmmonly used alternatives

are in vivo periodontal wound healing models.

A wide variety of different animal models have been used to facilitate

study of human periodontitis and its response to regenerative proœdures. The

most commonly used models employ rodents, dogs and non-human primates

(Page and Schroeder, 1982). The ability to closely replicate the periodontal

lesions of man and ultimately their utility as models for the study of human

periodontitis is a critical factor in model seledion. There are no aarently used

animal models of periodontitis that perfectly replicate human periodontal lesions;

indeed, it is uncertain if a perfect model will ever be found. Significant differences

between humans and animals in diets, oral habits, masticatory patterns, life-

span, tissue destruction pathways, tissue morphology, host defense mechanisms

and genetic traits underline the essential validity of this statement. Accordingly,

the diversity among animal species in susœptibility, progression, and

morphological features of periodontitis necessitate that the animal model used for

the research project be selected with great care and awareness of their

limitations.

The utilization of an in vivo model of the healing periodontium is often

complicated by the presence of bacteria and other soluble factors in the oral

cavity (e-g. salivary proteins, crevicular fluid enzymes). Consequently, the ability

to sequester experimentally these confounding variables can facilitate studies of

periodontal wound healing and simpfify the interpretation of biological outwmes.

A second and altical determinant is whether the healing wound can be

influenced by the protocol under test and whether experimentall y-induced

variations can be measured reliably. For example, critical size defeds, which do

not heal spontaneously during the lifetime of the animal, are very useful in

identifying the factors that promote wound healing since it is known that without

the experimental intervention, healing does not progress. Unfortunately, there are

no available models for critical sire defects in rodent periodontiurn. Indeed, King

et al. (1997) showed that despite variations in size, essentially al1 fenestration-

type defects created in rat molar periodontium will heal spontaneowly. As a

complete review of al1 animal models is beyond the swpe of this thesis, I will

briefly review three cornmonly used types of model systems that have been

employed in periodontal wound healing research.

1. Non-human primates

Monkeys, baboons and other non-human primates are models that

morphologically, most closely resemble the human dentition and periodontium.

With appropriate application of silk ligatures, the kinetics and microbiology of

disease progression can also be modeled reasonably well (Holt et al., 1988).

However, experiments using nonhuman primates are expansive, sample sizes

are generally small and ethical review boards very closely monitor the

experimental designs of non-human primate experiments. The position of

monkeys within the evolutionary hierarchy of animals and their dose physical

resemblance to man didates a careful consideration of experimental design, the

potential for animal suffering and the reasons for using them in experiments.

2. Dogs

Although they exhibit anatomical, topographical and physiological differences of

the periodontium campared to humans (Page and Schroeder, 1982). dogs

provide an excellent animal model to study gingivitis and other periadontal

diseases. Infiammatory lesions are readily induced by soft diets and several

breeds (0.g. beagles) spontaneously exhibit progressive periodontitis lesions with

increased age. The gingival lesion extends apically through the junctional

epithelium and the kinetics of increasing crevice depth are similar (but not

identical) to the deepening periodontal pocket in man and nonhuman primates

(Schroeder et al., 1975). Experimental periodontal regeneration in dogs has

produced results that are often difficult to interpret since some of the lesions heal

spontaneously, most notably in furcation defects (Bogle et al., 1 983). Further,

high purchase and maintenance costs are important considerations in the ability

of many investigators to obtain a substantial sample size.

3. Rodents

The teeth and periodontal tissues of rodents, such as mice, rats and hamsters,

undergo marked physiological changes throughout their relatively short life span

(Vignery and Baron, 1980; McCulloch and Melcher, 1983a). Even in teeth of

limited eruption such as the molars, any pathological changes must be

interpreted in the context of dynamic tissue remodelling that includes rapid matrix

turnover in bone, cementum and periodontal ligament. Another important

difference between rodents and man is that the sulwlar epithelium of rats (Page

and Sdiroeder, 1982) including the gen-free rat (Yamasaki et ai., 1979) is

keratinized compared to the non-keratinized sulwler epithelium of humans. This

feature may impact on the formation of pocket epithelium and the apical

extension of inflammatory cell infiltrates.

In spite of limitations related to variations in morphology and spontaneity

of healing in rats compared to humans, the rat periodontal window wound model

developed by Melcher (1 970) and further refined by Gould et al. (1977) and Lekic

et al. (1996b) provides an excellent system to study cell repopulation and cell

differentiation in the absence of oral bacteria and epithelial downgrowth. The

window wound model can be easily standardized and in the hands of

experienœd operators can provide relatively reproducible data with respect to

wound site, configuration and stability (Gould et al., 1 980; Lekic et al., 1996b).

While the rat model has several advantages, there are also several

drawbacks. First, the absence of bacterial biofilm formation perhaps over-

simplifies the wound healing environment since this important factor is eliminated

fmrn the model. Further, the window wound heals spontaneously over time, even

without therapeutic intervention. Nevertheless, the predictability and reliability of

the model facilitates studies of cell proliferation and cell differentiation in

response to various implanted materials on periodontal regeneration (Nguyen et

al., 1 997; King et al., 1 997, Rajshankar et al., 1 998). In this study, I have fowsed

on the impact of Emdogaina~ on cell differentiation in the repopulation response in

the wounded rat periodontal ligament. Consequently, the shortcomings of the

model described above (i.e. lack of bacterial biofilm formation, spontaneous

healing over time) do not significantly impact on the central outcomes of the

study.

II. Statement of the problem

Periodontal diseases are high prevalence infections that cause the destruction of

connective tissue, the loss of fibrous attachment and the resorption of alveolar

bone and cementum. If untreated, these infections can lead eventually to tooth

loss. Currently, most treatment approaches for the management of periodontitis

focus on the elimination of bacterial infection and the stabilization of the marginal

lesion. However, frequent consequences of periodontitis and of surgical

periodontal treatment include elongated dinical crowns and root exposure,

reduced periodontium and increased sensitivity to thermal stimuli. Accordingly,

despite the successes of conventional treatment, the ultimate goals of

periodontal therapy including the regeneration of connective tissue, the formation

of cementum and bone, and the attachment of new connective tissue fibers into

previously exposed root cementum (Egelberg, 1987, Aukhil et al., 1990, Polson,

1986) remain elusive.

One possible approach to enhance periodontal regeneration is to mirnic

the processes that take place dwing the development of the root and periodontal

tissues. Notably, Emdogaim is an enamel matrix derivative that may be able to

promote hard and soft tissue regeneration on the basis of its presumptive ability

to recapitulate critical events in tooth morphogenesis (Hammarstrom et al.,

1997). Indeed, some limited and very preliminary studies in the rat periodontal

window wound model have demonstrated that Emdogaim may produœ a large

increase in the volume of nascent bone and cementum matrices as early as one

week after wounding. Other data indicate that EmdogairS may greatly improve

the rate and nature of the regenerative process (Heijl et al., 1 997). Nevertheless,

separate studies have dernonstrated equivocal results following the topical

application of this agent (Sculean et al., 1 999. 2001 ).

Evidently, a deeper understanding of how Emdogaiw rnay promote

regeneration is essential for developing an improved biological basis for its use in

periodontal therapy, and for optirniring protocols that may lead to favorable and

predictable outwmes. Currently, the effects of Emdogaim on the differentiation

of cells in regenerating periodontal tissues are not known and the molecular

mechanisms by which this agent may prornote wound healing are poorly

understood. My hypothesis is that Emdogaiw facilitates regeneration of

periodontal tissues by promoting the differentiation of cells that are required for

the synthesis of new periodontal ligament, bone and cementum matrices.

To test my hypothesis, I have framed the following objectives to

investigate the efFed of Emdogaiw on the periodontium using the rat periodontal

window wound rnodel:

1-Ta compare the arnount of new bone and cementum as well as the

width of the periodontal ligament space following EmdogaiW or vehide

treatment of periodontal defects.

2-To study cellular differentiation following wounding and topical

application of Emdogaiw using intracellular and extracellular expression of

osteopontin, bon8 sialoprotein, osteocalcin, and a-smooth muscle actin as

markers of cellular differentiation.

III. Materials and Methods

Twenty-seven male CBL Wstar rats (90-115 g) were obtained from Charles

River mlmington, MA). The vehicle control (propylene glycol alginate) and

Emdogaim (stock concentration of 30 mglml) were obtained from BlORA AB

(Malmci, Sweden). '~pro l ine (specific activity=24 Cilmmol) at a final injected

concentration of 1 pCilg body weight was provided by Mandel Sdentific (Guelph,

ON). Mouse monoclonal antibodies to osteopontin (OPN; clone # MPIIIBI O) and

bone sialoprotein (BSP; Clone # WVIDl [9C5]) were obtained from the

Hybridoma Bank, Johns Hopkins University, Baltimore, MD. Mouse monoclonal

antibody to a-smooth muscle actin was obtained from Sigma Chemical (Clone #

lA4; Oakville, ON). Polydonal rabbit antibody to osteocatcin (OC) was obtained

from Dr. William Butler (University of Texas, Houston, Texas). Kodak nudear

track liquid emulsion (NTB-2) for radioautography was obtained from Kodak

(Eastman Kodak Co., Rochester, NY).

A. Wound model

The periodontal window wound model originally described by Melcher (1970) and

modified later by Gould et al. (1977) and Lekic et al. (1 996a,b) provides a

repopulating wound in which periodontal ligament cells are recruited to

regenerate spontaneously alveolar bone, cernenturn and periodontal ligament

(Le. non&tical size defect). The model facilitates studies of periodontal cellular

differentiation since a relatively well-syndwonized cohort of connective tissue

œfls proliferates and subsequently differentiates during the repopulation

response. Rats were caged in pairs in a room with a 12-hour darWlight cycle and

provided with food and water ad libitum. A total of 27 rats were included in these

experiments. Animals were anesthetized with Halothane (1.3%) and nitrous

0xide:oxygen (2:l). An incision -1 cm in length was made through the skin

overlying the incisor trunk. The posterior masseter muscle was identified and

retracted to locate the mental nerve that was dissected free and the anterior

fibers of the masseter muscle were incised at their point of insertion into the

mandible to expose the underlying bone. A rnodified end-aitting bur (0.6 mm in

diameter) driven by a slow speed dental hand-piece was used to drill a hole with

a final diameter of -0.8-1.0 mm through the alveolar bone over the mesiobuccal

root of the mandibular first molar. The hole was located mid-way between the

gingival rnargin and the mental nerve and was -1 mm posterior to the anterior

edge of the mandible. This hole extended to the most lateral surface of the

periodontal ligament but did not actually penetrate the soft tissue of the

periodontal ligament. W~th the aid of a dissecting microscope (Wild M3Z 10X)

and a 27 gauge needlq the periodontal ligament was extirpated dom to the level

of cernentum and the wound was cleared of debris with saline and a wet gauze.

Before the wound site was closed, either the vehide (control) or EmdogainQ54 at a

concentration of 3 mglml or 30 mglml (see below), was placed into the defect but

without overfiowing the defed. For al1 animals, wounds were performed on both

the left and the right mandibular first molars. Finally, the tissues were closed with

4 0 V i q î intempted sutures that resorbed spontaneously.

B. Preparation of implants and experimen fa1 aldesign

EmdogaiM was distributed in increments of -0.3 g each. The propylene glycol

alginate was prepared as 10 ml or 7 0 0 ml aliquots. An hour before the surgical

procedure was begun, the enamel matrix derivative was mixed with either 10 ml

or 100 ml of the propylene glycol alginate to yield an Emdogaiw preparation with

a concentration of 30 mglml or 3 mglml, respedively. The wound sites were

either: 1) implanted with the propylene glycol alginate (vehicle contrd); or 2)

implanted with EmdogainO at a concentration of 30 mglml; or 3) implanted with

EmdogairVB at a concentration of 3 mglml.

To study the effects of EmdogainQ3 on wound healing over time, three

animals (6 sides) for each experimental condition (vehicle control and

EmdogaiM3 at a concentration of 30 mglml or 3 rnglml) were sacrificed by CO2

asphyxiation at 7, 14 and 21 days following surgery. These time periods were

chosen on the basis of previous experiments (Lekic et al., 1996) showing that

these time periods correspond to the early proliferative stage of healing, to the

matrix formation stage of healing and finally to the completion of healing. One

and three days prior to sacrifice, one rat belonging to each of the three

experimental groups and from each of the three time periods studied, was

injected intraperitoneally with '~groline diluted with saline to produce a final

injectable concentration of 1 pCi/g body weight in a total volume of 2 ml. Injection

of the '~grol ine at 2 time periods facilitated assessrnent of the rate of matrix

synthesis in the healing tissues.

C. Tissue preparation

Following sacrifice, the mandible was removed, cleared of any attached soft

tissues and tnmmed at the mid-incisor and at the third rnolar region. Tissues

were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) at pH 7.4

for 24 hours at 4OC, demineralized for 24 hours in 0.2N HCI at rcom temperature

and washed in PBS at pH 7.4 for 24 hours, also at room temperature. Thereafter,

the specirnens were dehydrated by washing in graded ethanol solutions, cleared

in toluene and embedded in paraffin. Sections of -5 pm in thickness were cut

transversally to the longitudinal axis of the tooth with a Leitz microtome (mode1

#1512). Sections that were located closest to the drill site were stored on trays

and every fifteenth section was stained with toluidine Mue to determine the exact

location of the wound site. Sections in the middle of the wound sites were

attached ont0 Superfrost Plus@ slides (Fisher Scientific, Toronto, ON) and used

for immunohistochemical (Chen et al., 1991 b), morphornetric and

radioautographic analyses (Lekic et al., 1 996c).

D. lmmunohistochemistry

For immunohistochernical analyses, the sections were dewaxed in xylene (4 X

1.5 min) and rehydrated in a series of graded ethanol solutions (2 X 1.5 min in

100% ethanol, 1 X 2 min in 95% ethanol and 1 X 2 min in 70% ethanol). Slides

were washed in water (i X 5 min) as well as in PBS at pH 7.4 (2 X 5min). The

slides were subsequently incubated at room temperature with 3% H202 in

methanot for 30 min and protected from light. This step enabled inactivation of

endogenous peroxidase activity. Sections were washed with PBS at pH 7.4 (2 X

5 min).

To decrease non-specific background staining, sections were incubated

with a serumcontaining casein blocking solution (Rajshankar et al., 1998) for

one hour in a moist chamber, at room temperature. When staining for

osteopontin, bone sialoprotein and a-smooth muscle actin, mouse and horse pre-

immune sera were used at a dilution of 0.1% (vlv) in the blocking solution. For

osteocalcin staining, rabbit and goat pre-immune sera were used at a dilution of

0.02% (vfv) in the blocking solution (Vector, Burlingame. CA).

Sections were subsequently incubated at roorn temperature with one of

the following prirnary antibodies: mouse monoclonal antiosteopontin (1 : 1 Oûû),

mouse monoclonal anti-bone sialoprotein (1:500), rnouse monoclonal antia-

smooth muscle actin (1 :100) or rabbit polydonal antiosteocalcin antibody (150)

diluted with antibody diluting bMer (DAKO Diagnostics Laboratories,

Mississauga, ON) for 1.5 hours in a moist chamber. The sections were washed

with PBS at pH 7.4 (3 X 5 min) and incubated with secondary antibody diluted

with antibody diluting buffer (DAKO Diagnostics Laboratories. Mississauga, ON)

for 30 min in a moist chamber, also at room temperature. The secondary

antibody was biotinylated horse anti-mouse (1:2W) for osteopontin and bone

sialoprotein, and the same antibody was used at a dilution of 1 :A00 for staining of

a-smooth muscle actin. For osteocalcin, biotinylated goat anti-rabbit (1 50) was

used. Sections were washed with PBS at pH 7.4 (2 X Smin) and incubated at

room temperature with streptavidin horseradish peroxidase (PK-6100,

Vectastain) for 30 min, in a moist chamber. The sections were washed with PBS

at pH 7.4 (2 X 5 min) and incubated with diaminobenzidine (DAB) (SK-4100,

Vector, Burlingame, CA) for 15 min. The color reaction was stopped by gentle

rinsing with water ovemight. Finally, the sections were counter-stained with

hematoxylin and eosin, mounted in Penount, coverslipped and examined with a

Bioquant image analyzer (see below).

E. Radioautography

Radioautographs were prepared from the specimens labeled with 'H-proline

using the dipping method (Lekic et al., 1996~). First, slides were dewaxed in

xylene (4 X 1.5 min.) and rehydrated in a series of graded ethanol solutions (2 X

1.5 min in 10O0h ethanol, 1 X 2 min in 9S0h ethanol and 1 X 2 min in 70%

ethanol). The sections were air-dried for about one hour and dipped in Kodak

NTB-2 (Eastman Kodak Co., Rochester, NY), plaœd in light-proteded, dry boxes

and exposed for 2 weeks at 4OC. Following exposure, slides were developed for

6 minutes in D-19 developer (Eastman Kodak). This reaction was stopped by

placing the slides into 30°h ethanol for 30 sec and fixed for 5 min. The slides

were washed with water and stained with hematoxylin and eosin through the

emulsion.

F. MorphomeMc ana&ses

For specimens stained with hematoxylin and eosin and in sections

immunostained for osteopontin, several measurements of tissue structure were

made to assess the effect of Emdogaim on healing. Sections were examined

with a Leica Orthoplan microscope equipped with a drawing tube and analyzed

with a computerized morphornetry program (Bioquant). From each wound block,

three sections separated by at least 100 pm were examined. In each section, two

square sampling gnds of -40,000 were superimposed over the wound

compartment. First, the reversal line in the bone at the ait edges of the wound

was digitized in osteopontin-immunostained sections. This gave an estimate of

the original wound margins in the alveolar bone overlying the wound site. The

areas of new bone produœd within the defed were traced. These two areas

were then used to wmpute the percentage of new bone area fomed in the

wound. Next, the width of the periodontal ligament of wounded and contralateral

unwounded sites was measured and a ratio computed. This measurement

estimates whether possible growth of bone into the periodontal ligament space

(and hence loss of periodontal ligament homeostasis) has occurred. Finally, the

thickness of cementum on wounded and unwounded sides was deterrnined and

a ratio cornputed. The various measurements obtained from the wound side were

compared to the unwounded contralateral side of the rwt.

Analyses of immunostaining for intracellular Golgilendoplasmic retiwlum

proteins (OPN, BSP, OC or aSMA-stained cells) and for extracellular matrix

proteins (OPN, BSP or OC) were conducted in the regenerating bone

compartment of the wound by digital rnorphometric analyses (Lekic et al.,

l996a, b, 1 997) and by stereological procedures (McCulloch et ai., 1990).

Measurement of these proteins has been used to identify disaete stages in the

formation of bone in vivo (Chen et al., 1992; Yoon et al., 1 987) and for studies of

cell differentiation in periodontal tissues (Lekic et al., 1996b). The identification of

cells with intracellular immunostaining for these proteins can suggest the

presence of cells committed to the osteogenic or cementogenic lineages (Chen

et al., 1992.1993; McKee et al., 1 993) or to the fibroblastic lineage (for a-smooth

muscle actin; Arora and McCulloch, 1994). An intraocular grid system with 100

squares was superimposed over the wounded site to facilitate counting. Cells

with intracellular staining present within the gnd were wunted to provide

quantitative estimates of cell differentiation within the repopulating zone of the

PL. Total cell counts present within this grid were also obtained. These two cell

counts were then used to compute the percentage of cells with intracellular

staining for each marker present in the wound. Second, the presenœ or absence

of staining in the matrix for OPN, BSP or OC within each of the 100 squares of

the grid was counted and expressed as a %. This datum gives an estimate of the

volume density of nascent bone matrix produced by osteogenic cells. Previous

detailed threedimensional analyses of mineralized tissues have shown that

counting the number of cells that stain for a specific protein within a coherent grid

system allows the abundance of the cells present in a tissue to be estimated from

a 24imensional tissue section (McCulloch et al., 1990). Further, application of a

coherent grid system for estimating the relative abundance of staining for a

specific protein from a tissue section also gives an estimate of the perewitage of

the tissue volume that contains the protein (Rajshankar et al., 1998).

Analyses of radioautographs allowed measurement of matrix protein

synthesis rates by labeling nascent bone and cementum matrices (McCulloch

and Heersche 1988). In transverse sections, the distance between the two

labels, measured by morphometry, gives an estimate of matrix appositional rates

and of the extent of healing in the cementum and bone compartments. Grain

counts pet 1000 prd were also cornputed to provide an estimate of the

incorporation of labeled proline into matrix and these counts were adjusted by

subtracting background counts obtained over mature dentine.

G. Statistical analyses

Two-factor analysis of variance was performed to evaluate the differences

between the three dnig treatment groups over time after wounding. Further,

possible interactions between treatrnent and time were tested. The SAS system

(Cary, NC) was used to analyze the data. Analyses were wnducted for

measurements of tissue structure, immunostaining assessments and matrix

protein synthesis rates in the different sites examined. The data from each rat

(n=6) were pooled to obtain an animal-specific mean value. These animal means

were considered as single measures that were representative of each animal.

The results were reported as meanzstandard error of the mean where n-3

animals for the experimental groups and treated controls. Individual differences

between groups and time of sacrifice for the various experimental outcornes were

assessed by Tukey's test. A value of pc0.05 was considered statistically

significant.

IV. Results

A. ?%riodonial ligament homeostasis

The possible growth of bone into the periodontal ligament space and henœ the

loss of periodontal ligament homeostasis was assessed by measuring the width

of the periodontal ligament of both wounded and unwounded sides and by

computing the ratio of these widths. A ratio of greater than one indicates that the

periodontal ligament width of the wounded side is wider than that of the

unwounded side and may suggest ingrowth of osteogenic cells. At 7 days, rats

treated with either the vehicle control or Erndogairm at 3 mglml exhibited a

slightly wider periodontal ligament space than unwounded sites (Figure 1 A,B).

By 14 and 21 days, this ratio was very close to unity, indicating that homeostasis

of the periodontal ligament was restored. In uintrast, defects treated with

Emdogaiw ai 30 mglml showed widening of the periodontal ligament width at 7

days (p

Emôogmn 3 me(ml Emdogain 30 mglm1

Vehicle umtrol m Emdogain 3 mgiml rn EmQgain 30 mglml - --

74

m m (da- ' F

Figure 1.

Morphometric analyses.

A) lmmunohistochemical staining for osteopontin in vehicle control-treated

defect. Rat sacrificed at 14 days after wounding. Cementum (C), periodontal

ligament (PL) and bone (B). Note growth of bone into wound defect and

restoration of periodontal ligament width. Magnification 100X. 6 ) Histogram

showing ratio of periodontal ligament width of experimental (wounded)

defectslcantrol (non-wounded) sides. Defects were treated with vehicle control,

or EmdogaimB at 3 or 30 mglml. Rats were sacrifiœd at 7, 14 and 21 days after

wounding. Data are meanskSEM of ratios (n=6). *-pcO.001 wmpared to vehicle

control and to Emdogaim (3 mglml) at 7 days. C) Imrnunohistochemical staining

for bone sialoprotein on non-wounded side at 7 days after wounding. Cementum

(C), periodontal ligament (PL) and bone (B). Note pmminent staining for bone

sialoprotein in cementum. Magnification 4WX. D) Histogram showing ratio of

cementum thickness of experimental (wounded) defecWcontrol (non-wounded)

sides. Defects were treated with vehicle control, or EmdogaiM 3 or 30 mgtml.

Rats were sacrificed at days 7, 14 and 21. Data are meanstSEM (n=6).

*-p

B. Cernentum

The effect of Emdogairm on cementurn formation was deterrnined by measuring

the thickness of cementum on the wounded and unwounded sides and by

wmputing a ratio of these thicknesses. mus, the cementum thickness on the

wounded side was normalized to the relatively constant width of the cementum

on the unwounded side within each section. A ratio of less than one indicated

that the œmentum thickness on the wounded side is less than that of the

unwounded side and reflects loss of cementum at early time periods after

healing. This morphometric approach obviates systematic errors that may arise

because of section orientations deviating from a perpendicular orientation to the

longitudinal axis of the tooth. During wounding and extirpation of the periodontal

ligament, the cementum was scored with the drill and resorptian of cementum

ocairred as part of the healing process. Consequently, at 7 days, the ratio of the

cementum thickness was well below one for al1 groups. indicating that œmentum

formation was limited (Figure 1 C,D). As expected in normal periodontal wound

healing, the cementum thickness increased neady 2-fold between 7 and 14 days

(p

Figure 2.

Analyses of immunostaining for intracellular osteopontin and extracellular

matrix osteopontin.

A) Defect treated with Emdogaim (3 mglml) in rat sacfificecl at 7 days; cell with

intracellular staining for osteopontin (arrows) adjacent to cut bone margin of

wound defect. Magnification 400X. 6) Histogram showing perœntage of cells in

wound site staining for osteopontin/total cell count. Defects were treated with

vehicie control, or Erndogaiw at 3 or 30 mglml. Rats were sacrificed at 7, 14 and

21 days. Data are meanskSEM (n=6). C) Vehicfe control-treated defect in rat

sacrificed at 21 days showing extracellular staining for osteopontin (arrows) at

reversal lines. Magnification 250X. D) Histogram showing stereological analysis

of osteopontin staining in matrix. Defects were treated wWi vehicle control, or

Emdogaiw at 3 or 30 mglml. Rats were sacrificed at days 7, 14 and 21. Data

are means2SEM (n=6). - peO.01 comparing matrix staining for bone sialoprotein between 7 and 14 days.

while much lower effects were due to Emdogaim and even less to interanimal

variation. The coefficient of variation of the rnodel was 40%.

C. Bone

The area of new bone area fomed within the wound area was assessed by

digital morphometry of osteopontin-stained sections. Osteopontin-stained

reversal lines in the bone were used to identify the cut bone margin and

consequently, served to locate the original wound edges. Only limited bone

formation ocairred by 7 days after wounding in al1 3 groups (Figure 1 E,F). By 14

days, there was a dramatic IO-fold increase in bone area (p0.4)

at any of the three time periods. As shown by ANOVA, virtually al1 of the variation

in the bone measurements was attributable to time-dependent effects of healing

while there was virtually no effect because of Emdogaim, inter-animal variation

or interactions between time and dnig. The CO-efficient of variation of the ANOVA

model was 4 4 O h .

O. Osteogenic dHemntiaüon

lrnmunostaining for intracellular Golgilendoplasmic reticulum proteins and for

extracellular matrix proteins was conducted in the bone compartment of the

wound (Lekic et al., 1996a,b, 1997). The percentage of cells with intracellular

staining for osteopontin, a m a r k of early osteogenic differentiation, was sparse

at al1 time periods after wounding (-2%) and there was no difference in the % of

- . - . . - . - - - - -- . -. - . - - - - -- - . - - - - - - --- 0 Vehicle coritroi - 6 . œ EmdoQoin 3 mgiml c I Emdogain 30 mdml 1

Timm (dry.)

- -- O Vehicb cmtrd

100 r Emdogain 3 mgiml Y 0 . Emcbgain 30 mgiml - - - - -- - --

Figure 3.

Analyses of immunostaining for intracellular bone sialoprotein and

extracellular matrix bone sialoprotein.

A) Defect treated with Emdogaim at 30 mglml in rat sacrificed at 14 days after

wounding. Cell with intracellular staining for bone sialoprotein (arrow) in middle of

PL. Magnification 400X. B) Histogram of percentage of cells in wound site

staining for bone sialoproteinltotal cell count. Defects were treated with vehide

control, or Emdogaim at 3 or 30 mglml. Rats were sacrificed at 7, 14 and 21

days after wounding. Data are meanszSEM (n=6). *-pc0.05 wmparing defects

treated with Erndogaiw 3 and 30 mglml at 14 and 21 days after wounding. C)

Defect treated with Erndogaim at 30 mglml in rat sacrificed at 14 days after

wounding showing extracellular staining for bone sialoprotein (arrows) in nascent

bone matrix. Magnification 250X. D) Histogram of stereological analysis of bone

sialoprotein staining in matrix. Defects were treated with vehicle wntrol, or

Emdogaiw at 3 or 30 mglml. Rats were sacrificed at 7, 14 and 21 days after

wounding. Data are meanstSEM (n=6). -- pcO.01 comparing rnatrix staining for bone sialoprotein between 7 and 14 days; *-pe0.05 comparing defects treated

with Emdogaim 3 and 30 mglml at 7 and 21 days.

celfs with intracellular osteopontin staining between the three treatment groups at

any of the three time points (Figure 2 48). Stereological methods were also

used to estimate the volume density of nascent matrix in the wound site that was

imrnunostained for osteopontin. As expected. the time after wounding strongly

affected the expression of osteopontin in the matrix and there was a 5-fold

increase of osteopontin-stained matrix between 7-14 days (pc0.01) but there was

no difference between Emdogain treatments. 8y 21 days, -80% of th8 matrix

was stained with osteopontin. However. Emdogaim. even at 30 mglml, exerted

no detectable effect on extracellular osteopontin staining at al1 three time points

(Figure 2 D).

A small % of cells (1-4%) exhibited intracellular staining for bone

sialoprotein. a marker of the minerakation stage of osteogenic differentiation. At

14 and 21 days. there were apparent differences between the vehicle control-

treated defeds and the defeds treated with Emdogaiiwp at 3 and 30 mglml

(Figure 3 A,B) but these d-ifferences were small and of marginal statistical

significance (p=0.05). For extracellular bone sialoprotein staining in nascent

matrix, there was a nearly 4-fold increase of staining between 7-1 4 days (p

-. - - - - -. - - . - - -

6 . )Emdogain 3 mglml m Emdonein 30 mdml

Figure 4.

Analyses of immunostaining for intracellular osteocalcin and extracellular

matrix osteocalcin.

A) Defect treated with Emdogaim at 3 mglml; rat sacrificed at 21 days showing

an osteocyte with intracellular staining for osteocalcin (arrow). Magnification

400X. B) Histogram of percentage of cells in wound site staining for

osteocalcinltotal cell count. Defects were treated with vehide control or

Emdogain@ at 3 or 30 mglml. Rats were sacrifiiced at 7, 14 and 21 days after

wounding. Data are means5SEM (n=6). *-p'-0.01 comparing defects treated with

vehicle control or Emdogaiw (30 mglml) at day 7. C) Defect in vehicle control-

treated rat saaificed at 21 days depicting extracellular staining for osteocalcin

(arrows) in wound defect. Magnification 250X. D) Histogram showing

stereological analysis of osteocalcin staining in matrix within the wound site.

Defects were treated with vehicle control or Emdogaim at 3 or 30 mglml. Rats

were sacrificed at 7, 14 and 21 days after wounding. Data are meanszSEM

(n=6). l p 4 . 0 1 comparing defects treated with EmdogainQB (30 mglml) at day 7.

for osteocalcin was small (0.53%) for al1 sampling periods (Figure 4 A,B) and

most of these stained cells appeared to be osteocytes. There were some

variations in the % of osteocatcin-positive cells in the different groups at the

various time points of sampling. At 7 days after wounding, there was a very low

% of immunostained cells in the Emdogaim 3 mglml group (p

Time (da*

Figure 5.

Analyses of immunostaining for intracellular a-smooth muscle actin.

A) Defect treated with Emdogaim (3 mglml) in rat sacrificed at 14 days after

wounding. Cementum (C) and bone (B). Fibroblasts (F) and blood vessels (BV)

stain for a-smooth muscle actin. Magnification 4ûOX. B) Histogram of percentage

of cells in wound site staining for a-smooth muscle actiriltotal cell count. Defects

were treated with vehicle cuntrol or Emdogaim at 3 or 30 mglml. Rats were

sacrificed at 7, 14 and 21 days after wounding. Data are rneanszSEM (n=6). ' pe0.05 camparing intracellular staining for a-smooth muscle actin between 7 and

21 days after wounding.

significantly by 21 days after wounding for al1 groups (